The objective of the proposed studies is to develop understanding of the mechanism(s) of retroviral genome selection and packaging. Over the past 31/2years, we (i) developed an NMR approach for structural studies of relatively large RNAs, (ii) determined the structure of a monomeric form of the 101-nucleotide core encapsidation RNA element (WCES) of the Moloney Murine Leukemia Virus (MLV) packaging signal, both free in solution and bound to its cognate nucleocapsid (NC) protein; and (iii) demonstrated that NC binding is regulated by an RNA conformational switch, providing a novel mechanism that explains how MLV may specifically package a diploid genome. In concurrent studies, we identified elements within the genomes of the human immunodeficiency (HIV) and Rous sarcoma (RSV) viruses that bind their cognate NC proteins with high affinity. Preliminary NMR studies suggest that genome recognition by these retroviruses involves substantially different RNA structural elements and protein-RNA interactions. Having completed studies of several isolated protein-RNA components that promote packaging, we now intend to study larger fragments of retroviral genomes to better understand how multiple components interact to collectively promote efficient packaging. Studies will initially focus on the native MLV (WCES)2 dimer (>200 nucleotides) and its complex with NC, the 80-nucleotide RSV ^Wpackaging element, and fragments of the HIV-1 packaging signal containing putative long-range interactions. In what represents a significant new direction, we will also study intact retroviral 5'-UTRs using a novel approach that involves NMR analysis of segmentally labeled RNAs coupled with in situ NMR-monitored partial enzymatic hydrolysis. These studies will address structural questions that have not been answered by chemical accessibility or mass spectrometric experiments, and should allow unambiguous identification of the RNA secondary structures that promote dimerization and NC binding. Major goals will be to test our MLV RNA switch hypothesis and determine if HIV or RSV utilize similar RNA switch mechanisms to specifically package diploid genomes. Studies of larger RNAs are technically challenging, but the potential payoff is high and should ultimately lead to detailed models of intact retroviral 5'-UTR and protein:UTR structures that direct genome packaging and retrovirus assembly. Such knowledge should not only facilitate the development of better approaches for the treatment of AIDS and cancer, but also assist in the development of more effective retrovirus-based vectors for the therapeutic delivery of corrective human genes.